More than one hundred billion neurons exist in human brain to form complex neural circuits. Only prescribed numbers of them are formed in the adequate positions as development progresses. These neurons have very complicated shapes which never be seen in other somatic cells and extend two kinds of processes dendrite and axon from a cell body which is protoplasm including a nucleus. A dendrite comprises numerous thorn structures called spine and forms postsynaptic region that has a function for receiving information from other cells. It is known that this neuron specific shape is determined by a neuron specific actin-binding protein.
On the other hand, brain is an important organ that controls not only the action at unconsciousness level but also what is called higher-order function such as emotion, memory, learning, and creation. However, it has not revealed yet how the regions in brain are determined and how the differentiation of brains that is specific in each region are occurred. Neuronal migration is essential for construction of brain tissue, for example in cerebral cortex, a layer structure is formed by division of neural stem cells (radial glial cell) at ventricular zone and radial migration thereof with the help of the radial processes inherited in division. Although it has been indicated that molecules such as PS-NCAM or Slit are involved in these migrations of neurons, the relation has been hardly revealed yet.
As aforementioned, radial migration of postmitotic neurons is essential for neocortical development (J. Comp. Neurol. 145, 61-83, 1972, Nat. Neurosci. 4, 143-150, 2001, Nature 409, 714-720, 2001). Neurons generated in the ventricular zone have to make at least two important decisions in order to reach their destination correctly: when to start and where to stop migration. The stop of migration is thought to be regulated by Reelin (Nature 374, 719-723, 1995, Nature 389, 730-733, 1997, Nature 389, 733-737, 1997, Neuron 24, 471-479, 1999, Neuron 24, 481-489, 1999, Cell 99, 635-647, 1999, Cell 97, 689-701, 1999, Neuron 27, 33-44, 2000), however, the molecule relating to the start of migration has been poorly understood. An exception has been reported that disruption of an actin-binding protein Filamin 1 results in a human neuronal migration disorder, periventricular nodular heterotopia, in which many neurons remain lining the ventricular surface (Neuron 16, 77-87, 1996, Neuron 21, 1315-1325, 1998).
The present invention relates to a protein of the effects of controlling cell migration and cell death of such as neurons and a DNA encoding the protein, particularly, an object of the present invention is to provide a method of controlling cell migration and/or cell death and a method of screening a promoter or an inhibitor of the effects of controlling cell migration and/or cell death with the use of proteins controlling the cell motility and cell death of neurons and the DNA encoding the proteins by interacting an actin-binding protein and promoting the degradation of the actin-binding protein.
Analysis of the cerebral cortex having disorder in layer structure is thought to provide an important clue for clarification of molecular mechanism relating to neuronal migration during the development of cerebral cortex, for instance the clarification of molecular mechanism which arrests cell migration is progressing rapidly by the study of reeler mouse. Likewise, periventricular nodular heterotopia, in which immovable neurons remain at neuroepithelial layer is thought to be another clue for solving the mechanism for starting/maintaining the migration of neurons, and abnormality of an actin-binding protein Filamin 1 has been revealed to be a cause. (Though “Filamin 1” is sometimes called “Filamin A”, it is indicated “Filamin 1” in the present invention.)
Meanwhile, the inventors reported about a rat nascent stage cerebral cortex-derived cytoskelton-associated novel protein FILIP (Filamin-interacting protein), it was predicted that the FILIP (S-FILIP) molecule comprised 965 amino acid residues in total, and revealed that it comprised coiled-coil structure including leucine zipper motifs at N-terminal-half of the molecule. Moreover, yeast two-hybrid screening or immunoprecipitaion analyses revealed that the C-terminal-half of FILIP molecule is combined with an actin-binding protein, Filamin 1. Filamin 1 is an essential molecule for cell migration during cerebral cortex formation period, and it is known that mutation of Filamin 1 gene causes periventricular nodular heterotopia characterized in migration disorder of cerebral cortical neuron. This led to the possibility that FILIP (S-FILIP) controls cell migration by associating with Filamin 1 to control those function at developing cerebral cortex. To verify this hypothesis, FILIP was expressed in a cultured cell and the aspect of cell migration was observed with time following. In consequence, migration of FILIP-expressing cell was controlled compared to the control, FILIP (S-FILIP) was indicated as a negative control factor of cell migration.
Subsequently, as the result of a keen study by the present inventors, FILIPs (L-FILIP and S-FILIP) were identified, FILIPs were found that they had functions for controlling cell motility and cell death, and the present invention was completed. That is, FILIP molecule (965 amino acid residues; S-FILIP; GenBank accession number D87257) (SEQ ID NOS: 3 and 4 in sequence listing) and L-FILIP which comprises 1212 residues, being constructed by adding 247 residues to molecule on the N terminal side (GenBank accession number AB055759) (SEQ ID NOS: 1 and 2 in the sequence listing).
Moreover, the result of a further study by the present inventor, human FILIP molecule (1213 amino acid residues; h-FILIP; -GenBank accession number AB086011) (SEQ ID NOS: 5 and 6 in the sequence listing), which is a human orthologue of mouse L-FILIP, was identified from human DNA library.
The present inventors found that when the novel protein L-FILIP or S-FILIP was introduced into cells, these molecules partially coexisted with filamentous-actin within the cells, and in the same cell, the degradation of filamentous-actin was yielded, it became smaller and shorter, the lamellipodia formation ratio from cell membrane was decreased, and the cell migration ratio was significantly decreased. They also found that L-FILIP which is a novel molecule had more significant Filamin 1 degradation promoting effect as well as it expressed more protein at cerebral cortex neuroepithelium than S-FILIP, from the result of investigation using cultured cells. These facts revealed that S-FILIP but L-FILIP mainly plays the role of controlling cell migration negatively by promoting degradation of Filamin 1 at cerebral cortex neuroepithelium.
When S-FILIP or L-FILIP and Filamin 1 were expressed in the same cell, the change in Filamin 1 was observed, and the degradation of Filamin 1 progressed by expression of FILIP was observed similarly as aforementioned. These changes were also significant at L-FILIP. When the expression of Filamin 1 at the brain of normal rats during their fatal stage was examined, expression of Filamin 1 gene was observed, while a number of cells were observed of which expression amount of Filamin 1 protein had largely decreased in cells localized in ventricular zone, where expression of FILIP gene being observed, and cell migration toward cortical plate having not yet occurred. On the other hand, reduction of the cell number was identified in the cultured cell to which novel molecule L-FILIP was introduced, and it was revealed that FILIPs were also related to the control of cell death. The present invention was completed based upon the knowledge mentioned above.